Method of screening antiplatelet

ABSTRACT

A screening tool for an antiplatelet agent wherein the tool is a human ADP receptor P2T AC  protein, a variation functionally equivalent thereto, or a homologous protein thereof, and a screening tool for an antiplatelet agent wherein the tool is a transformant which is transformed with an expression vector comprising a DNA encoding the protein and is expressing the polypeptide are disclosed. Further, a method for detecting an ADP receptor P2T AC  ligand, antagonist, or agonist by using the screening tool for an antiplatelet agent, and a method for screening an antiplatelet agent by the detecting method are disclosed.

TECHNICAL FIELD

[0001] The present invention relates to a method for screeningantiplatelet agents.

BACKGROUND ART

[0002] The platelet was discovered in 1842 by Donne [C. R. Acad.Sci.(paris), 14, 336-368, 1842], and has long been regarded as acomponent of blood necessary for hemostasis. It is now known that theplatelet plays not only a main role in the hemostatic system, but alsomultifunctional roles, for example, a clinically notable generation ofarteriosclerosis, circulatory diseases including thrombotic diseases,metastasis of cancer, inflammation, rejection after grafting,participation in immune reaction, or the like.

[0003] At present, revascularization by pharmacological or physicalmethods is carried out to treat thrombotic diseases and ischemicdiseases. However, it has been recently found that the activation,adhesion, and/or aggregation of the platelets is promoted by a collapseof blood vessel tissue including an endothelial cell afterrevascularization, or a collapse of a fibrinolysis-coagulation balancecaused by a medicament per se, which becomes a clinical problem. Forexample, it is known that, after revascularization by a thrombolytictherapy using t-PA or the like, the fibrinolytic activity and/orcoagulative activity are activated, and then the systemicfibrinolysis-coagulation balance collapses. Clinically, this causesre-occlusion, and becomes a critical problem therapeutically (J. Am.Coll. Cardiol. 12, 616-623, 1988).

[0004] In addition, the PTCA (Percutaneous transluminal coronaryangioplasty) therapy has quickly become widely used, and has achievedgood results in the treatment of diseases based on aortostenosis orcoronary stenosis such as angina, myocardial infarction, or the like.However, the therapy injures blood vessel tissue including anendothelial cell, and acute coronary obstruction, and restenosis, whichis observed in approximately 30% of cases, become a problem.

[0005] The platelet plays an important role in these various thromboticdisorders (such as re-occlusion or the like) after the revascularizationtherapy. Therefore, an antiplatelet agent is desired as an agent fortreating or preventing these thrombotic disorders.

[0006] In this connection, adenosine 5′-diphosphate (ADP) is known as animportant factor which induces or promotes the activation, adhesion, andaggregation of the platelets. ADP is released from platelets activatedby collagen, thrombin, or the like, or from hemocytes, vascularendothelial cells, or organs injured by revascularization or the like.It is considered that ADP activates the platelets via a Gprotein-coupled ADP receptor P2T located in the platelet membrane(Biochem. J., 336, 513-523, 1998).

[0007] It has been suggested that a platelet ADP receptor P2T_(PLC)which is coupled to Gq, one of the G proteins, and increases anintracellular Ca²⁺ concentration via phospholipase C (PLC), and aplatelet ADP receptor P2T_(AC) which is coupled to Gi, one of the Gproteins, and suppresses an activity of adenylate cyclase (AC) arepresent as platelet ADP receptors. At present, the platelet ADP receptorP2T_(PLC) has been identified as the receptor known as platelet ADPreceptor P2Y1, but the entity of platelet ADP receptor P2T_(AC) is notidentified (Kunapuli, S. P. et al., Trends Pharmacol. Sci., 19, 391-394,1998).

[0008] It is considered that Ticlopidine or Clopidogrel used as anantiplatelet agent functions by inhibiting the ADP receptor P2T_(AC) viaits metabolite in a body (Savi, P. J., Pharmaclo. Exp. Ther., 269,772-777, 1994). Further, ARL67085, which is synthesized as a derivativeof adenosine triphosphate (ATP), which is an ADP receptor antagonist ina body, exhibits an activity of suppressing a platelet aggregation bythe antagonist activity against the platelet ADP receptor P2T_(AC), andthe effectiveness thereof is proven by using a thrombosis model (Mills,D. C., Thromb. Hemost., 76, 835-856, 1996; and Humphries, R. G., TrendsPharmacol. Sci., 16, 179-181, 1995). Further, a derivative of Ap4A[P¹,P⁴-di(adenosine-5′)tetraphosphate] exhibits an activity suppressingthe platelet aggregation by ADP, by the antagonist activity against theplatelet ADP receptor P2T_(AC), and the effectiveness thereof is provenby using a thrombosis model (Kim, B. K., Proc. Natl. Acad. Sci. USA, 89,2370-2373, 1992).

[0009] From the above information, an antagonist against the plateletADP receptor P2T_(AC) is desired as a strong antiplatelet agent.However, Ticlopidine or Clopidogrel exhibits a weak antiplateletactivity, and has problems such as a strong side effect or the like.Further, ARL67085 or derivatives thereof (ATP analogues), derivatives ofAp4A, or the like, which is studied as the ADP receptor antagonist, is aderivative of nucleotide, and then an oral bioavailability is notsufficient, and further problems arise such as a weak activity ofsuppressing the platelet aggregation. Therefore, an ADP receptorantagonist having a strong oral bioavailability is intensely desired(CAPRIE STEERING COMMITTEE, Lancet, 348, 1329-1339, 1996).

[0010] However, the ADP receptor P2T_(AC) protein has not beenidentified as yet. Therefore, it is difficult to construct a convenientsystem for screening such a compound, and farther, the development ofthe ADP receptor P2T_(AC) antagonist has not progressed.

[0011] In this connection, with regard to a DNA encoding a polypeptideconsisting of the same amino acid sequence as that of a human ADPreceptor P2T_(AC) protein, which may be used in the present invention,and an amino acid sequence deduced from the DNA, there are severalreports (WO00/22131, WO00/31258, WO00/28028, and WO98/50549 pamphlets).However, ligands are not elucidated in the reports, and no reportsdisclose that the protein is an ADP receptor located in the platelet.

DISCLOSURE OF INVENTION

[0012] Therefore, the object of the present invention is to provide aconvenient screening system to obtain an adenosine diphosphate (ADP)receptor P2T_(AC) antagonist which is useful as an antiplatelet agent,and a novel antiplatelet agent.

[0013] With the aim of solving the aforementioned problems, the presentinventors have conducted intensive studies and, as a result,successfully isolated a nucleic acid (more particularly, an HORK3 gene)encoding the P2T_(AC) receptor, and have determined the nucleotidesequence thereof and the deduced amino acid sequence. Further, theinventors prepared a vector comprising the nucleic acid encoding thereceptor, and a host cell comprising the vector, and made it possible toproduce a novel recombinant P2T_(AC) receptor by expressing the P2T_(AC)receptor by the use of the host cell. The inventors confirmed that thereceptor exhibited an ADP receptor P2T_(AC) activity, and thus thereceptor and the cell expressing the receptor could be used as ascreening tool for an antiplatelet agent. The inventors established amethod for detecting whether or not a test compound is an ADP receptorP2T_(AC) ligand, antagonist, or agonist by using the receptor or thecell expressing the receptor, and a method for screening an antiplateletagent by using the detecting method. The inventors confirmed thatcompounds known to exhibit an antiplatelet activity (more particularly2MeSAMP or AR-C69931MX) exhibited an antagonist activity of thereceptor, by using the detecting method, and showed that an antagonistof the receptor was certainly useful as an antiplatelet agent. Further,the inventors established a process for manufacturing a pharmaceuticalcomposition for antiplatelet, comprising the detecting step, andcompleted the present invention.

[0014] Namely, the present invention relates to:

[0015] [1] a screening tool for an antiplatelet agent, wherein the toolis

[0016] (1) a polypeptide (hereinafter sometimes referred to as “humanADP receptor P2T_(AC) protein”) having an amino acid sequence of SEQ IDNO: 2, or

[0017] (2) a polypeptide (hereinafter referred to as “variationfunctionally equivalent”) having an amino acid sequence in which one orplural amino acids are deleted, substituted, and/or added at one orplural positions in an amino acid sequence of SEQ ID NO: 2, andexhibiting an activity (hereinafter referred to as “ADP receptorP2T_(AC) activity”) of suppressing an adenylate cyclase activity bybinding to ADP and coupling with Gi:

[0018] [2] a screening tool for an antiplatelet agent, wherein the toolis a polypeptide (hereinafter referred to as “homologous protein”)having an amino acid sequence having a 90% or more homology with anamino acid sequence of SEQ ID NO: 2, and exhibiting an activity ofsuppressing an adenylate cyclase activity by binding to ADP and couplingwith Gi (hereinafter the screening tools of the items [1] and [2] for anantiplatelet agent are collectively referred to as “polypeptide-typescreening tool for an antiplatelet agent”);

[0019] [3] a screening tool for an antiplatelet agent, wherein the toolis a transformant which is transformed with an expression vectorcomprising a DNA encoding (1) a polypeptide having an amino acidsequence of SEQ ID NO: 2 (i.e., human ADP receptor P2T_(AC) protein) or(2) a polypeptide having an amino acid sequence in which one or pluralamino acids are deleted, substituted, and/or added at one or pluralpositions in an amino acid sequence of SEQ ID NO: 2, and exhibiting anactivity of suppressing an adenylate cyclase activity by binding to ADPand coupling with Gi (i.e., variation functionally equivalent), and isexpressing the polypeptide;

[0020] [4] a screening tool for an antiplatelet agent, wherein the toolis a transformant which is transformed with an expression vectorcomprising a DNA encoding a polypeptide having an amino acid sequencehaving a 90% or more homology with an amino acid sequence of SEQ ID NO:2, and exhibiting an activity of suppressing an adenylate cyclaseactivity by binding to ADP and coupling with Gi (i.e., homologousprotein), and is expressing the polypeptide (hereinafter the screeningtools of the items [3] and [4] for an antiplatelet agent arecollectively referred to as “transformant-type screening tool for anantiplatelet agent”);

[0021] [5] a method for detecting whether or not a compound to be testedis an ADP receptor P2T_(AC) ligand, comprising the steps of:

[0022] bringing a polypeptide of the item [1] or [2] (i.e., the humanADP receptor P2T_(AC) protein, variation functionally equivalent, orhomologous protein), a cell membrane fraction comprising thepolypeptide, or a transformant of the item [3] or [4] into contact withthe compound to be tested, in the presence of a labeled ligand of an ADPreceptor P2T_(AC), and

[0023] analyzing a change of an amount of the labeled ligand which bindsto the polypeptide, the cell membrane fraction, or the transformant(hereinafter referred to as “ligand detecting method”);

[0024] [6] a method for detecting whether or not a compound to be testedis an ADP receptor P2T_(AC) antagonist or agonist, comprising the stepsof:

[0025] bringing the compound to be tested into contact with atransformant of the item [3] or [4] which is co-expressing a G proteinchimera comprising a polypeptide fragment having an activity promoting aphospholipase C activity of a G protein promoting the phospholipase Cactivity and a polypeptide fragment having a receptor-coupled activityof Gi, said G protein chimera having an amino acid sequence of SEQ IDNO: 11 at the C-terminus; and

[0026] analyzing a change of an intracellular Ca²⁺ concentration in thetransformant (hereinafter referred to as “Ca²⁺-type detecting method”);

[0027] [7] a method for detecting whether or not a compound to be testedis an ADP receptor P2T_(AC) antagonist or agonist, comprising the stepsof:

[0028] bringing a transformant of the item [3] or [4] into contact withthe compound to be tested, in the presence of a platelet ADP receptorP2T_(AC) agonist, and

[0029] analyzing a change of an intracellular cAMP concentration in thetransformant (hereinafter referred to as “cAMP-type detecting method”);

[0030] [8] a method for screening an antiplatelet agent, comprising thesteps of:

[0031] detecting whether or not a compound to be tested is an ADPreceptor P2T_(AC) ligand, antagonist, or agonist by the ligand detectingmethod, the Ca²⁺-type detecting method, or the cAMP-type detectingmethod, or a combination thereof, and selecting the ADP receptorP2T_(AC) antagonist; and

[0032] [9] a process for manufacturing a pharmaceutical composition forantiplatelet, comprising the steps of:

[0033] detecting whether or not a compound to be tested is an ADPreceptor P2T_(AC) ligand, antagonist, or agonist by the ligand detectingmethod, the Ca²⁺-type detecting method, or the cAMP-type detectingmethod, or a combination thereof, and preparing a medicament.

[0034] The “Gi” is a subfamily of the G proteins which are coupled to areceptor and function as a signal transduction and amplification factorinto a cell, and a G protein which suppresses an adenylate cyclaseactivity. When the adenylate cyclase activity is suppressed, forexample, an intracellular cAMP concentration decreases.

[0035] The “G protein promoting the phospholipase C activity” is asubfamily of G proteins which are coupled to a receptor and function asa signal transduction and amplification factor into a cell, and a Gprotein which promotes a phospholipase C activity. When thephospholipase C activity is induced, for example, an intracellular Ca²⁺concentration increases. As the G protein promoting the phospholipase Cactivity, there may be mentioned, for example, Gq.

[0036] The “ADP receptor P2T_(AC) ” means a polypeptide having the ADPreceptor P2T_(AC).

BRIEF DESCRIPTION OF DRAWINGS

[0037]FIG. 1 is a graph showing the effect of 2MeSAMP in an ADP-inducedplatelet aggregation of platelet-rich plasma (PRP) derived from humanblood-treated with sodium citrate.

[0038]FIG. 2 is a graph showing the effect of AR-C69931MX in anADP-induced platelet aggregation of PRP derived from human blood-treatedwith sodium citrate.

BEST MODE FOR CARRYING OUT THE INVENTION

[0039] The present invention will be explained in detail hereinafter.

[0040] (1) The Screening Tool for an Antiplatelet Agent

[0041] The screening tool of the present invention for an antiplateletagent includes a polypeptide-type screening tool for an antiplateletagent and a transformant-type screening tool for an antiplatelet agent.

[0042] 1) The polypeptide-type screening tool for an antiplatelet agent

[0043] The polypeptide-type screening tool of the present invention foran antiplatelet agent includes

[0044] (i) a screening tool for an antiplatelet agent, wherein the toolis the human ADP receptor P2T_(AC) protein, i.e., the polypeptide havingthe amino acid sequence of SEQ ID NO: 2;

[0045] (ii) a screening tool for an antiplatelet agent, wherein the toolis a variation functionally equivalent, i.e., a polypeptide having anamino acid sequence in which one or plural amino acids are deleted,substituted, and/or added at one or plural positions in the amino acidsequence of SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC)activity; and

[0046] (iii) a screening tool for an antiplatelet agent, wherein thetool is a homologous protein, i.e., a polypeptide having an amino acidsequence having a 90% or more homology with the amino acid sequence ofSEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC) activity.

[0047] The polypeptide having the amino acid sequence of SEQ ID NO: 2,which may be used as the polypeptide-type screening tool of the presentinvention for an antiplatelet agent, is a human ADP receptor P2T_(AC)protein consisting of 342 amino acid residues. Although it has beensuggested that the ADP receptor P2T_(AC) protein exists in platelets,the entity thereof had not been identified until the present inventorsfirst elucidated its function.

[0048] Then, it was first published in a reference after the prioritydate of the present application that the polypeptide having the aminoacid sequence of SEQ ID NO: 2 is a platelet ADP receptor [Nature, 409,Jan. 11, 2001, 202-207; J. B. C., 276, (11), March 16, 8608-8615, 2001;WO01/46454 pamphlet]. In the basic patent application of the priority ofthe WO01/46454 pamphlet, although a rat ADP receptor gene was cloned andthe sequence thereof was determined, as to a human ADP receptor gene,only a clone containing a partial sequence was obtained and the sequencethereof was determined. Therefore, it was first accomplished by thepresent applicant that the human ADP receptor P2T_(AC) protein and thepolynucleotide encoding the protein was obtained, the function thereofwas elucidated, and a patent application relating to the invention ofthe method for screening an antiplatelet agent using them was filed.

[0049] The variation functionally equivalent which may be used as thepolypeptide-type screening tool of the present invention for anantiplatelet agent is not particularly limited, so long as it is apolypeptide having an amino acid sequence in which one or plural(preferably 1 to 10, more preferably 1 to 5) amino acids are deleted,substituted, and/or added at one or plural positions in the amino acidsequence of SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC)activity. Further, an origin of the variation functionally equivalent isnot limited to a human.

[0050] The variation functionally equivalent includes, for example,human variations of the human ADP receptor P2T_(AC), and ADP receptorsP2T_(AC) derived from organisms other than a human (such as a mouse, arat, a hamster, or a dog) or variations thereof, and further proteinsobtained by artificially modifying these native proteins (i.e., humanvariations, or ADP receptors P2T_(AC) derived from organisms other thana human or variations thereof) or the human ADP receptor P2T_(AC) bygenetic engineering techniques. The term “variation” as used hereinmeans an individual difference between the same proteins in the samespecies or a difference between homologous proteins in several species.

[0051] Human variations of the human ADP receptor P2T_(AC), or ADPreceptors P2T_(AC) derived from organisms other than a human orvariations thereof may be obtained by those skilled in the art based onthe information of a nucleotide sequence (for example, the nucleotidesequence of SEQ ID NO: 1) of the human ADP receptor P2T_(AC) gene. Inthis connection, genetic engineering techniques may be performed inaccordance with known methods (for example, Maniatis, T. et al.,“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY, 1982, or the like), unless otherwise specified.

[0052] For example, an appropriate probe or appropriate primers aredesigned in accordance with the information of a base sequence of thehuman ADP receptor P2T_(AC) gene. A PCR method or a hybridization methodis carried out using a sample (for example, total RNA or an mRNAfraction, a cDNA library, or a phage library) derived from an organism(for example, a mammal such as a human, a mouse, a rat, a hamster, or adog) of interest and the primers or the probe to obtain a gene encodingthe protein. A desired protein can be obtained by expressing theresulting gene in an appropriate expression system and confirming thatthe expressed protein suppresses the adenylate cyclase activity bybinding to ADP and coupling with Gi, for example, by the methoddescribed in Example 3 or 4.

[0053] Further, the protein artificially modified by genetic engineeringtechniques can be obtained by, for example, the following procedure. Agene encoding the protein is obtained by a conventional method such assite-specific mutagenesis (Mark, D. F. et al., Proc. Natl. Acad. Sci.USA, 81, 5662-5666, 1984). A desired protein can be obtained byexpressing the resulting gene in an appropriate expression system andconfirming that the expressed protein suppresses the adenylate cyclaseactivity by binding to ADP and coupling with Gi, for example, by themethod described in Example 3 or 4.

[0054] The homologous protein which may be used as the polypeptide-typescreening tool of the present invention for an antiplatelet agent is notparticularly limited, so long as it is a polypeptide having an aminoacid sequence having a 90% or more homology with the amino acid sequenceof SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC) activity. Thehomologous protein may have an amino acid sequence having preferably a95% or more homology, more preferably a 98% or more homology, mostpreferably a 99% or more homology, with respect to the amino acidsequence of SEQ ID NO: 2.

[0055] The term “homology” as used herein means a value which can beobtained by a BLAST package [Basic local alignment search tool;Altschul, S. F. et al., J. Mol. Biol., 215, 403-410, (1990)]. Thehomology in the amino acid sequence can be calculated by a BLAST searchalgorithm. More particularly, this value can be obtained by using abl2seq program (Tatiana A. Tatusova and Thomas L. Madden, FEMSMicrobiol. Lett., 174, 247-250, 1999) with a default parameter in aBLAST package (sgi32bit edition, version 2.0.12; obtained from NCBI).Default parameters of the bl2seq program include “blastp” as a searchprogram, “0” as a cost to open a gap, “0” as a cost to extend a gap,“SEG” as a filter of the query sequence, and “BLOSUM62” as a matrix.

[0056] The polypeptides which may be used as the polypeptide-typescreening tool of the present invention for an antiplatelet agent (i.e.,the human ADP receptor P2T_(AC) protein, the variations functionallyequivalent, and the homologous proteins; hereinafter referred to as“polypeptide for a screening tool”) may be obtained by various knownmethods, such as known genetic engineering techniques using a geneencoding a protein of interest. More particularly, the polypeptide for ascreening tool may be prepared by culturing a transformant describedbelow (i.e., a transformant which is transformed with an expressionvector comprising a DNA encoding the polypeptide for a screening tooland expressing the polypeptide) under a condition in which an expressionof the polypeptide for a screening tool may be performed, and separatingand purifying the protein of interest from the resulting culture bycommonly used methods for a separation and a purification of receptorproteins.

[0057] When the polypeptide for a screening tool is prepared, the methodfor obtaining the gene encoding the polypeptide is not particularlylimited. For example, when the human ADP receptor P2T_(AC) protein isprepared, for example, the DNA consisting of the nucleotide sequence ofSEQ ID NO: 1 may be used as the gene encoding the protein. In thisconnection, codons for each amino acid are known and can be optionallyselected and determined by the conventional method, for example, bytaking a codon usage of each host to be used into consideration(Crantham, R. et al., Nucleic Acids Res., 9, r43-r74, 1981).

[0058] The DNA consisting of the nucleotide sequence of SEQ ID NO: 1 maybe obtained by, for example, ligating DNA fragments prepared by achemical synthesis method, or a polymerase chain reaction (PCR) method(Saiki, R. K. et al., Science, 239, 487-491, 1988) using a cDNA libraryderived from a cell or tissue capable of producing the human ADPreceptor P2T_(AC) protein as a template and an appropriate primer setdesigned in accordance with the nucleotide sequence of SEQ ID NO: 1. Asthe cell or tissue capable of producing the human ADP receptor P2T_(AC)protein, there may be mentioned, for example, human platelets, the humanbrain, or the like. As the primer set, there may be mentioned, forexample, a combination of an oligonucleotide consisting of thenucleotide sequence of SEQ ID NO: 3 and an oligonucleotide consisting ofthe nucleotide sequence of SEQ ID NO: 4.

[0059] A separation and purification method which may be used forpreparing the polypeptide for a screening tool is not particularlylimited, but can be performed, for example, in accordance with thefollowing procedure. For example, a cell membrane fraction containingthe polypeptide for a screening tool can be obtained by culturing cellsexpressing the polypeptide for a screening tool on the surface thereof,suspending the cultured cells in a buffer, homogenizing the suspension,and centrifuging the homogenate. After the resulting cell membranefraction is solubilized, the polypeptide for a screening tool can bepurified by treating the mixture with a commonly used treatment with aprotein precipitant, ultrafiltration, various liquid chromatographytechniques such as molecular sieve chromatography (gel filtration),adsorption chromatography, ion exchange chromatography, affinitychromatography, or high performance liquid chromatography (HPLC), ordialysis, or a combination thereof. In this connection, when the cellmembrane fraction is solubilized, characteristics of the receptor can bemaintained after the solubilization, by using as mild as possible asolubilizing agent (such as CHAPS, Triton X-100, digitonin or the like).

[0060] When the polypeptide for a screening tool is prepared, aconfirmation of the expression of the polypeptide, a confirmation ofintracellular localization thereof, a purification thereof, or the likemay be easily carried out by expressing the polypeptide for a screeningtool as a fusion protein with an appropriate marker sequence in frame,if necessary. As the marker sequence, there may be mentioned, forexample, a FLAG epitope, a hexa-histidine tag, a hemagglutinin tag, amyc epitope, or the like. Further, by inserting a specific sequencerecognized by a protease such as enterokinase, factor Xa, thrombin, orthe like between the marker sequence and the polypeptide for a screeningtool, the marker sequence may be removed by the protease. For example,there is a report in which a muscarinic acetylcholine receptor and ahexa-histidine tag were connected by a thrombin recognition sequence(Hayashi, M. K. and Haga, T., J. Biochem., 120, 1232-1238, 1996).

[0061] 2) The transformant-type screening tool for an antiplatelet agent

[0062] The transformant-type screening tool of the present invention foran antiplatelet agent includes

[0063] (i) a screening tool for an antiplatelet agent, wherein the toolis a transformant which is transformed with an expression vectorcomprising a DNA encoding the human ADP receptor P2T_(AC) protein and isexpressing the polypeptide;

[0064] (ii) a screening tool for an antiplatelet agent, wherein the toolis a transformant which is transformed with an expression vectorcomprising a DNA encoding the variation functionally equivalent and isexpressing the polypeptide; and

[0065] (iii) a screening tool for an antiplatelet agent, wherein thetool is a transformant which is transformed with an expression vectorcomprising a DNA encoding the homologous protein and is expressing thepolypeptide.

[0066] A host cell which may be used for preparing the transformants(hereinafter referred to as “transformant for a screening tool”) whichmay be used as the transformant-type screening tool of the presentinvention for an antiplatelet agent is not particularly limited, so longas the polypeptide for a screening tool can be expressed. As the hostcell, there may be mentioned, for example, commonly used knownmicroorganisms, such as Escherichia coli or Saccharomyces cerevisiae, orknown cultured cells, such as vertebral cells (for example, a CHO cell,HEK293 cell, or COS cell) or insect cells (for example, an Sf9 cell). Asthe vertebral cell, there may be mentioned, for example, a COS cell as asimian cell (Gluzman, Y., Cell, 23, 175-182, 1981), a dihydrofolatereductase defective strain of a Chinese hamster ovary cell (CHO)(Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA, 77,4216-4220, 1980), a human embryonic kidney derived HEK293 cell, or a293-EBNA cell (Invitrogen) obtained by introducing an EBNA-1 gene ofEpstein Barr Virus into the HEK293 cell.

[0067] An expression vector which may be used for preparing thetransformant for a screening tool is not particularly limited, so longas the polypeptide for a screening tool can be expressed. An appropriateexpression vector can be selected in accordance with a host cell to beused.

[0068] As an expression vector for a vertebral cell, a vector containinga promoter positioned upstream of a gene to be expressed, an RNAsplicing site, a polyadenylation site, a transcription terminationsequence, and the like may be generally used. The vector may furthercontain a replication origin, if necessary. As the expression vector,there may be mentioned, for example, pSV2dhfr containing an SV40 earlypromoter (Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981),pEF-BOS containing a human elongation factor promoter (Mizushima, S. andNagata, S., Nucleic Acids Res., 18,5322, 1990), pCEP4 containing acytomegalovirus promoter (Invitrogen), or the like.

[0069] More particularly, when the COS cell is used as the host cell, avector having an SV40 replication origin, capable of performing anautonomous replication in the COS cell, and having a transcriptionpromoter, a transcription termination signal, and an RNA splicing site,may be used as the expression vector. As the vector, there may bementioned, for example, pME18S (Maruyama, K. and Takebe, Y., Med.Immunol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and Nagata, S.,Nucleic Acids Res., 18, 5322, 1990), pCDM8 (Seed, B., Nature, 329,840-842, 1987), or the like.

[0070] The expression vector may be incorporated into COS cells by, forexample, a DEAE-dextran method (Luthman, H. and Magnusson, G., NucleicAcids Res., 11, 1295-1308, 1983), a calcium phosphate-DNAco-precipitation method (Graham, F. L. and van der Ed, A. J., Virology,52, 456-457, 1973), a method using a cationic liposome reagent(Lipofectamine; Gibco BRL), an electroporation method (Neumann, E. etal., EMBO J., 1, 841-845, 1982), or the like.

[0071] When the CHO cell is used as the host cell, a transformantcapable of stably producing the polypeptide for a screening tool can beobtained by carrying out a co-transfection of an expression vectorcomprising the DNA encoding the polypeptide for a screening tool,together with a vector capable of expressing a neo gene which functionsas a G418 resistance marker, such as pRSVneo (Sambrook, J. et al.,“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY, 1989), pSV2-neo (Southern, P. J. and Berg, P., J. Mol. Appl. Genet.,1, 327-341,1982), or the like, and selecting a G418 resistant colony.

[0072] When the 293-EBNA cell is used as the host cell, for example,pCEP4 (Invitrogen) containing a replication origin of Epstein Barr Virusand capable of performing an autonomous replication in the 293-EBNAcell, or the like may be used as the expression vector.

[0073] The transformant for a screening tool may be cultured inaccordance with a conventional method, and the polypeptide for ascreening tool is produced in the cell or on the cell surface. As amedium to be used in the culturing, a medium commonly used in a selectedhost cell may be appropriately selected. For example, in the case of theCOS cell, for example, a medium such as an RPMI-1640 medium, aDulbecco's modified Eagle's minimum essential medium (DMEM), or the likemay be used, by supplementing it with a serum component such as fetalbovine serum (FBS) or the like if necessary. In the case of the 293-EBNAcell, a medium such as a Dulbecco's modified Eagle's minimum essentialmedium (DMEM) or the like with a serum component such as fetal bovineserum (FBS) or the like and G418 may be used.

[0074] The transformant for a screening tool is not particularlylimited, so long as the polypeptide for a screening tool is expressed.As the transformant for a screening tool, it is preferable to express aG protein in which the amino acid sequence at the C-terminus is that ofSEQ ID NO: 11 (Asp-Cys-Gly-Leu-Phe), in addition to the polypeptide fora screening tool. The amino acid sequence of SEQ ID NO: 11 is thatconsisting of five amino acid residues at the C-terminus of Gi.Hereinafter the “G protein in which the amino acid sequence at theC-terminus is that of SEQ ID NO: 11” will be referred to as “C-terminusGi-type G protein”.

[0075] As the C-terminus Gi-type G protein, there may be mentioned, forexample,

[0076] (1) Gi, or

[0077] (2) a G protein chimera which comprises a polypeptide fragmenthaving the activity promoting a phospholipase C activity of a G protein(such as Gq) promoting the phospholipase C activity and a polypeptidefragment having a receptor-coupled activity of Gi, and which further hasan amino acid sequence of SEQ ID NO: 11 at the C-terminus. Hereinafter,the G protein chimera comprising a polypeptide fragment having theactivity of promoting a phospholipase C activity and a polypeptidefragment having a receptor-coupled activity of Gi will be referred to asGqi.

[0078] The polypeptide for a screening tool recognizes the amino acidsequence consisting of five amino acid residues at the C-terminus of Gi(i.e., the amino acid sequence of SEQ ID NO: 11), and binds to Gi.Therefore, the polypeptide for a screening tool may be bound to not onlyGi, but also Gqi. When the polypeptide for a screening tool and theC-terminus Gi-type G protein are expressed in the transformant for ascreening tool, these polypeptides can bind to each other in the cell.

[0079] The “polypeptide fragment having the activity of promoting aphospholipase C activity of a Gq” is not particularly limited, so longas it does not comprise a C-terminal amino acid sequence which isnecessary to bind to a Gq-coupled platelet ADP receptor P2T_(PLC), andexhibits the activity of promoting the phospholipase C activity. Theremay be mentioned, for example, a polypeptide fragment at the N-terminalside of Gq in which the amino acid sequence consisting of five aminoacid residues at the C-terminus is deleted.

[0080] The “polypeptide fragment having a receptor-coupled activity ofGi” is not particularly limited, so long as it comprises the amino acidsequence consisting of five amino acid residues at the C-terminus of Gi,and does not exhibit an activity suppressing the adenylate cyclaseactivity. There may be mentioned, for example, a polypeptide fragment atthe C-terminal side of Gi, consisting of the amino acid sequence of SEQID NO: 11.

[0081] (2) The Detecting Method for an ADP Receptor P2T_(AC) Ligand,Antagonist, or Agonist

[0082] It can be detected whether or not a test compound is an ADPreceptor P2T_(AC) ligand, antagonist, or agonist, by using thepolypeptide for a screening tool or the transformant for a screeningtool as a detecting tool.

[0083] The method of the present invention for detecting whether or nota test compound is an ADP receptor P2T_(AC) ligand, antagonist, oragonist includes

[0084] 1) a method for detecting whether or not a test compound is aligand against the platelet ADP receptor P2T_(AC) (i.e., liganddetecting method);

[0085] 2) a method for detecting whether or not a test compound is anantagonist or agonist against the platelet ADP receptor P2T_(AC) , bythe use of changes of an intracellular Ca²⁺ concentration in thetransformant as an indicator (i.e., Ca²⁺-type detecting method);

[0086] 3) a method for detecting whether or not a test compound is anantagonist or agonist against the platelet ADP receptor P2T_(AC), by theuse of changes of an intracellular cAMP content in the transformant asan indicator (i.e., cAMP-type detecting method); and

[0087] 4) a method for detecting whether or not a test compound is anantagonist or agonist against the platelet ADP receptor P2T_(AC), by theuse of a GTPγS binding method (i.e., GTPγS binding-type detectingmethod).

[0088] These detecting methods will be explained in this orderhereinafter.

[0089] 1) The ligand detecting method

[0090] The ligand detecting method of the present invention is notparticularly limited, so long as

[0091] (i) the human ADP receptor P2T_(AC) protein, variationfunctionally equivalent, or homologous protein (hereinafter referred toas “polypeptide for a detecting tool”), a cell membrane fractioncomprising the polypeptide for a detecting tool, or a transformantexpressing the polypeptide for a detecting tool (hereinafter thepolypeptide for a detecting tool, the cell membrane fraction, and thetransformant are collectively referred to as “ligand-detecting tool”)and

[0092] (ii) a labeled ligand are used. It may be carried out, forexample, in accordance with the following procedure.

[0093] The ligand-detecting tool is prepared. Assay conditions (forexample, a buffer to be used and the concentration thereof, ions to beadded to the buffer and the concentration thereof, and the pH in theassay system) are optimized. The ligand-detecting tool and a labeledligand are incubated in the optimized buffer, together with a testcompound, for a predetermined time. As the labeled ligand, for example,[³H] 2-methylthio-ADP (2MeSADP) may be used. After the reaction, thewhole is filtered with a glass filter or the like, and the filter iswashed with an appropriate volume of the buffer. The remainingradioactivity on the filter is measured by a liquid scintillationcounter or the like. It may be detected whether or not the test compoundis a ligand against the ADP receptor P2T_(AC) , by the obtained bindinginhibition of the radioactive ligand as an indicator. More particularly,when the remaining radioactivity on the filter in the presence of thetest compound is lower than that in the absence of the test compound, itmay be decided that the test compound is a ligand against the ADPreceptor P2T_(AC) . It may be carried out, for example, under theconditions described in Example 6.

[0094] 2) The Ca²⁺-type detecting method

[0095] In the Ca²⁺-type detecting method of the present invention, atransformant (hereinafter referred to as transformant for Ca²⁺-typedetection) co-expressing

[0096] (i) the polypeptide for a detecting tool and

[0097] (ii) a G protein chimera (such as Gqi) which comprises apolypeptide fragment having the activity promoting a phospholipase Cactivity of a G protein promoting the phospholipase C activity and apolypeptide fragment having a receptor-coupled activity of Gi, and whichfurther has an amino acid sequence of SEQ ID NO: 11 at the C-terminus isused as the transformant. As to the transformant, it is preferable touse a cell in which an intracellular Ca²⁺ concentration is not increasedby ADP, as a host cell before the transformation. As the host cell,there may be mentioned, for example, C6-15, one of rat glioma cell lines(Change, K. et al., J. Biol. Chem., 270, 26152-26158, 1995).

[0098] In the case of detecting whether or not a test compound is anagonist in the Ca²⁺-type detecting method of the present invention, thetransformant for Ca²⁺-type detection is brought into contact with a testcompound, and then changes of the intracellular Ca²⁺ concentration inthe transformant for Ca²⁺-type detection are analyzed (i.e., measured ordetected) directly or indirectly. Changes of the Ca²⁺ concentration maybe, for example, directly analyzed by the use of a calcium-bindingfluorescence reagent (such as fura2, fluo3, or the like), or indirectlyanalyzed by analyzing a transcriptional activity of a gene [such as agene obtained by introducing an activator protein 1 (AP1) responsivesequence upstream of a luciferase gene] in which a regulation of thetranscription is dependent on the Ca²⁺ concentration.

[0099] When the transformant for Ca²⁺-type detection is brought intocontact with a test compound, and then the intracellular Ca²⁺concentration therein increases, it may be decided that the testcompound is an agonist against the ADP receptor P2T_(AC) . In thisconnection, as a control, a similar procedure is carried out using acontrol transformant not expressing the polypeptide for a detecting toolbut expressing Gqi, or a host cell before the transformation, instead ofthe transformant for Ca²⁺-type detection co-expressing the polypeptidefor a detecting tool and Gqi, and then it is preferable to confirm thatthe intracellular Ca²⁺ concentration in the control transformant or hostcell is not increased by the test compound.

[0100] In the case of detecting whether or not a test compound is anantagonist in the Ca²⁺-type detecting method of the present invention,the transformant for Ca²⁺-type detection is brought into contact with atest compound in the presence of a platelet ADP receptor P2T_(AC)agonist (such as 2MeSADP or ADP), and then changes of the intracellularCa²⁺ concentration therein are analyzed (i.e., measured or detected)directly or indirectly. When the transformant for Ca²⁺-type detection isbrought into contact with a test compound in the presence of a plateletADP receptor P2T_(AC) agonist, and then the increase in theintracellular Ca²⁺ concentration therein by the agonist is inhibited orsuppressed by the test compound, it may be decided that the testcompound is an antagonist against the ADP receptor P2T_(AC). This may becarried out, for example, under the conditions described in Example 3.

[0101] In this connection, as a control, the transformant for Ca²⁺detection is brought into contact with the platelet ADP receptorP2T_(AC) agonist in the absence of a test compound, and then it isnecessary to confirm a degree of the increase in the intracellular Ca²⁺concentration therein by the agonist.

[0102] As described above, in the Ca²⁺-type detecting method of thepresent invention, Gi per se is not used as the coupled protein, but Gqiis used. Therefore, it is possible to detect whether or not a testcompound is an antagonist or agonist by analyzing the Ca²⁺concentration, not the cAMP concentration. In general, measurement canbe carried out more easily and rapidly by using the Ca²⁺ concentration,in comparison with the cAMP concentration.

[0103] 3) The cAMP-type detecting method

[0104] In the cAMP-type detecting method of the present invention, atransformant expressing the polypeptide for a detecting tool(hereinafter referred to as “transformant for cAMP-type detection”) isused as the transformant. Gi is constitutively expressed in a commonlyused host cell, and thus the transformant for cAMP-type detection may beobtained by transforming a host cell with an expression vectorcomprising a DNA encoding the polypeptide for a detecting tool. As tothe transformant, it is preferable to use a cell in which anintracellular cAMP concentration is not decreased by ADP, as a host cellbefore the transformation. As the host cell, there may be mentioned, forexample, a CHO cell.

[0105] In the case of detecting whether or not a test compound is anagonist in the cAMP-type detecting method of the present invention, thetransformant for cAMP-type detection is brought into contact with a testcompound, and then changes of the intracellular cAMP concentration inthe transformant for cAMP-type detection are analyzed (i.e., measured ordetected) directly or indirectly. It is preferable that the transformantfor cAMP-type detection is brought into contact with a test compound inthe presence of a compound (such as forskolin) capable of increasing thecAMP concentration.

[0106] Changes of the cAMP concentration may be, for example, directlyanalyzed by the use of a commercially available cAMP measuring kit(Amersham or the like), or indirectly analyzed by analyzing atranscriptional activity of a gene [such as a gene obtained byintroducing a cAMP responsive element (CRE) upstream of a luciferasegene] in which a regulation of the transcription is dependent on thecAMP concentration.

[0107] When the transformant for cAMP-type detection is brought intocontact with a test compound, and then the intracellular cAMPconcentration therein decreases, it may be decided that the testcompound is an agonist against the ADP receptor P2T_(AC). In this case,the decrease in the cAMP concentration by a test compound may be easilydecided, when a compound (such as forskolin) capable of increasing thecAMP concentration coexists. In this connection, as a control, a similarprocedure is carried out using a host cell not expressing thepolypeptide for a detecting tool, instead of the transformant forcAMP-type detection expressing the polypeptide for a detecting tool andGi, and then it is preferable to confirm that the intracellular cAMPconcentration in the host cell is not decreased by the test compound.

[0108] In the case of detecting whether or not a test compound is anantagonist in the cAMP-type detecting method of the present invention,the transformant for cAMP-type detection is brought into contact with atest compound in the presence of a platelet ADP receptor P2T_(AC)agonist (such as 2MeSADP or ADP), and then changes of the intracellularcAMP concentration therein are analyzed (i.e., measured or detected)directly or indirectly. When the transformant for cAMP-type detection isbrought into contact with a test compound in the presence of a plateletADP receptor P2T_(AC) agonist (such as 2MeSADP or ADP), and then thedecrease in the intracellular cAMP concentration therein by the agonistis inhibited or suppressed by the test compound, it may be decided thatthe test compound is an antagonist against the ADP receptor P2T_(AC). Inthis case, the change of the cAMP concentration by a test compound maybe easily decided, when a compound (such as forskolin) capable ofincreasing the cAMP concentration coexists. It may be carried out, forexample, under the conditions described in Example 4 or 5.

[0109] Further, as a control, the transformant for cAMP detection isbrought into contact with the platelet ADP receptor P2T_(AC) agonist inthe absence of a test compound, and then it is necessary to confirm adegree of the decrease in the intracellular cAMP concentration thereinby the agonist.

[0110] 4) The GTPγS binding-type detecting method

[0111] In the GTPγS binding-type detecting method of the presentinvention, it may be detected whether or not a test compound is anantagonist or agonist against the platelet ADP receptor P2T_(AC) by aGTPγS binding method (Lazareno, S. and Birdsall, N. J. M., Br. J.Pharmacol., 109, 1120-1127, 1993), by using the polypeptide for adetecting tool, a cell membrane fraction comprising the polypeptide, ora transformant expressing the polypeptide, for example, in accordancewith the following procedure.

[0112] A cell membrane expressing the polypeptide for a detecting toolis mixed with ³⁵S-labeled GTPγS (400 pmol/L) in a mixed solution of 20mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 10 mmol/L MgCl₂, and 50 mmol/LGDP. After incubating in the presence of a test compound, and in theabsence of the test compound, each reaction liquid is filtered by usinga glass filter or the like, and then the radioactivity of remainingGTPγS on the filter is measured by using a liquid scintillation counteror the like. It may be detected whether or not the test compound is anagonist against the ADP receptor P2T_(AC) by using the specific increaseof the GTPγS binding in the presence of the test compound as anindicator. Further, it may be detected whether or not the test compoundis an antagonist against the ADP receptor P2T_(AC) by using thesuppression in the increase of the GTPγS binding by a platelet ADPreceptor P2T_(AC) ligand (such as 2MeSADP or ADP) in the presence of thetest compound, as an indicator.

[0113] (3) The Screening Method for an Antiplatelet Agent

[0114] A ligand, antagonist, or agonist against the platelet ADPreceptor P2T_(AC) may be screened by using the screening tool (includingboth the polypeptide-type screening tool for an antiplatelet agent andthe transformant-type screening tool for an antiplatelet agent) of thepresent invention for an antiplatelet agent.

[0115] As previously described, ADP is known as an important factorwhich induces or promotes activation, adhesion, and aggregation of theplatelet. Further, it is considered that ADP activates the platelet viaa G protein-coupled ADP receptor P2T located in the platelet membrane(Biochem. J., 336, 513-523, 1998). Furthermore, it is considered thatTiclopidine or Clopidogrel, which is a known antiplatelet agent,functions by inhibiting the ADP receptor P2T_(AC) via its metabolite ina body (Savi, P. J., Pharmaclo. Exp. Ther., 269, 772-777, 1994). It isshown that ARL67085 or derivatives thereof (ATP analogues), derivativesof Ap4A [P¹,P⁴-di(adenosine-5′)tetraphosphate], or the like exhibit anactivity suppressing the platelet aggregation by ADP, by the P2T_(AC)antagonist activity (Mills, D. C., Thromb. Hemost., 76, 835-856, 1996;Humphries, R. G., Trends Pharmacol. Sci., 16, 179-181, 1995; and Kim, B.K., Proc. Natl. Acad. Sci. USA, 89, 2370-2373, 1992). As shown inExample 7, ADP receptor P2T_(AC) antagonists (such as 2MeSAMP orAR-C69931MX) exhibit the antiplatelet activity.

[0116] From the above information, the ligand or antagonist against theplatelet ADP receptor P2T_(AC) is useful as a substance capable ofcontrolling the activation, adhesion, and aggregation of the platelet.Therefore, the above-mentioned polypeptide for a screening tool per se,or the transformant for a screening tool per se may be used forscreening an antiplatelet agent. Namely, the polypeptide for a screeningtool per se, or the transformant for a screening tool per se may be usedfor a screening tool.

[0117] Compounds to be tested which may be screened by using thescreening tool of the present invention for an antiplatelet agent arenot particularly limited, but there may be mentioned, for example,various known compounds (including peptides) registered in chemicalfiles, compounds obtained by combinatorial chemistry techniques(Terrett, N. K. et al., Tetrahedron, 51, 8135-8137, 1995), or randompeptides prepared by employing a phage display method (Felici, F. etal., J. Mol. Biol., 222, 301-310, 1991) or the like. In addition,culture supernatants of microorganisms, natural components derived fromplants or marine organisms, or animal tissue extracts may be used as thetest compounds for screening. Further, compounds (including peptides)obtained by chemically or biologically modifying compounds (includingpeptides) selected by the screening tool of the present invention for anantiplatelet agent may be used.

[0118] The screening methods of the present invention may be mainlydivided into the following four groups in accordance with the detectingmethods to be used. By using any one of the detecting methods, or acombination thereof, a substance which is useful as an antiplateletagent can be screened by detecting whether or not a test compound is aligand, antagonist, or agonist against the ADP receptor P2T_(AC), andthen selecting an antagonist among test compounds. The screening methodsof the present invention, i.e.,

[0119] 1) a method for screening a ligand against the platelet ADPreceptor P2T_(AC) (hereinafter referred to as ligand screening method);

[0120] 2) a method for screening an antagonist or agonist against theplatelet ADP receptor P2T_(AC) by the use of changes of an intracellularCa²⁺ concentration in the transformant as an indicator (hereinafterreferred to as Ca²⁺-type screening method);

[0121] 3) a method for screening an antagonist or agonist against theplatelet ADP receptor P2T_(AC) by the use of changes of an intracellularcAMP concentration in the transformant as an indicator (hereinafterreferred to as cAMP-type screening method); and

[0122] 4) a method for screening an antagonist or agonist against theplatelet ADP receptor P2T_(AC) by the use of a GTPγS binding method(hereinafter referred to as GTPγS binding-type screening method) will beexplained in this order hereinafter.

[0123] 1) The ligand screening method

[0124] The ligand screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

[0125] detecting whether or not a test compound is an ADP receptorP2T_(AC) ligand by using the ligand detecting method of the presentinvention, and

[0126] selecting the ADP receptor P2T_(AC) ligand.

[0127] An ADP receptor P2T_(AC) ligand can be screened by the use of thebinding inhibition of the radioactive ligand obtained by the liganddetecting method as an indicator. For example, a test compound isreacted under the conditions described in Example 6 for a predeterminedtime, and then a test compound in which IC₅₀ is 10 μM or less(preferably 1 μM or less) may be selected as a ligand by the use of thebinding inhibition of [³H]-2MeSADP as an indicator.

[0128] A substance useful as an antiplatelet agent may be screened byapplying the ligand which was screened by the ligand screening method ofthe present invention to the following Ca²⁺-type screening method,cAMP-type screening method, and/or GTPγS binding-type screening method,and then selecting an antagonist.

[0129] 2) The Ca²⁺-type screening method

[0130] The Ca²⁺-type screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

[0131] detecting whether or not a test compound is an ADP receptorP2T_(AC) antagonist or agonist by using the Ca²⁺-type detecting methodof the present invention, and

[0132] selecting the ADP receptor P2T_(AC) antagonist or agonist.

[0133] An agonist can be screened by the use of the increase of anintracellular Ca²⁺ concentration by a test compound in the transformantfor Ca²⁺-type detection as an indicator in the Ca²⁺-type detectingmethod.

[0134] An antagonist can be screened by an indicator in which theincrease of an intracellular Ca²⁺ concentration in the transformant forCa²⁺-type detection by a platelet ADP receptor P2T_(AC) agonist (such as2MeSADP or ADP) is inhibited or suppressed by a test compound in theCa²⁺-type detecting method.

[0135] For example, a test compound is reacted under the conditionsdescribed in Example 3 for a predetermined time, and then a testcompound in which IC₅₀ is 10 μM or less (preferably 1 μM or less) may beselected as a substance exhibiting the antagonist activity by the use ofinhibition of the increase in an intracellular Ca²⁺ concentration by2MeSADP or ADP as an indicator.

[0136] A substance useful as an antiplatelet agent may be screened byscreening an antagonist by using the Ca²⁺-type screening method of thepresent invention.

[0137] 3) The cAMP-type screening method

[0138] The cAMP-type screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

[0139] detecting whether or not a test compound is an ADP receptorP2T_(AC) antagonist or agonist by using the cAMP-type detecting methodof the present invention, and

[0140] selecting the ADP receptor P2T_(AC) antagonist or agonist.

[0141] An agonist can be screened by the use of the decrease of anintracellular cAMP concentration by a test compound in the transformantfor cAMP-type detection as an indicator in the cAMP-type detectingmethod.

[0142] An antagonist can be screened by an indicator in which thedecrease of an intracellular cAMP concentration in the transformant forcAMP-type detection by a platelet ADP receptor P2T_(AC) agonist (such as2MeSADP or ADP) is inhibited or suppressed by a test compound in thecAMP-type detecting method.

[0143] For example, a test compound is reacted under the conditionsdescribed in Example 4 or 5 for a predetermined time, and then a testcompound in which IC₅₀ is 10 μM or less (preferably 1 μM or less) may beselected as a substance exhibiting the antagonist activity by the use ofinhibition of the decrease in an intracellular cAMP concentration by2MeSADP or ADP as an indicator.

[0144] A substance useful as an antiplatelet agent may be screened byscreening an antagonist by using the cAMP-type screening method of thepresent invention.

[0145] 4) The GTPγS binding-type screening method

[0146] The GTPγS binding-type screening method of the present inventionis not particularly limited, so long as it comprises the steps of:

[0147] detecting whether or not a test compound is an ADP receptorP2T_(Ac) antagonist or agonist by using the GTPγS binding-type detectingmethod of the present invention, and

[0148] selecting the ADP receptor P2T_(AC) antagonist or agonist.

[0149] An agonist can be screened by the use of the increase of thespecific GTPγS binding by a test compound as an indicator in the GTPγSbinding-type detecting method.

[0150] An antagonist can be screened by an indicator in which theincrease of the GTPγS binding by a platelet ADP receptor P2T_(AC)agonist (such as 2MeSADP or ADP) is suppressed by a test compound.

[0151] A substance useful as an antiplatelet agent may be screened byscreening an antagonist by using the GTPγS binding-type screening methodof the present invention.

[0152] (4) Manufacture of the Pharmaceutical Composition forAntiplatelet

[0153] The present invention includes an antiplatelet agent containing,as an active ingredient, an ADP receptor P2T_(AC) antagonist (forexample, compounds, peptides, antibodies, or antibody fragments)selected by the screening methods 2) to 4) or by a combination of thescreening methods 1) or 4).

[0154] It may be confirmed whether the selected ADP receptor P2T_(AC)antagonist exhibits the antiplatelet activity by detecting theinhibiting activity of human platelet aggregation, for example, by themethod described in Example 7.

[0155] Further, the present invention includes a method formanufacturing a pharmaceutical composition for antiplatelet comprisingthe steps of:

[0156] detecting, in a quality control test of a pharmaceuticalcomposition for antiplatelet, whether or not it is an ADP receptorP2T_(AC) ligand, antagonist, or agonist by the ligand detecting method,Ca²⁺-type detecting method, cAMP-type detecting method, and/or GTPγSbinding-type detecting method, and

[0157] preparing a medicament.

[0158] The preparation of the present invention containing the ADPreceptor P2T_(AC) antagonist (for example, compounds, peptides,antibodies, or antibody fragments) as an active ingredient may beprepared using carriers, fillers, and/or other additives generally usedin the preparation of medicaments, in accordance with the activeingredient.

[0159] Examples of administration include oral administration bytablets, pills, capsules, granules, fine granules, powders, oralsolutions and the like, and parenteral administration by injections(e.g., intravenous, intramuscular, or the like), suppositories,transdermal preparations, transmucosal absorption preparations and thelike. Particularly, in the case of peptides which are digested in thestomach, a parenteral administration such as intravenous injection orthe like, or preparation techniques in which the polypeptide is notdigested, such as a preparation technique disclosed in the WO95/28963pamphlet, is preferable.

[0160] In the solid composition for use in the oral administration, oneor more active substances may be mixed with at least one inert diluentsuch as lactose, mannitol, glucose, microcrystalline cellulose,hydroxypropylcellulose, starch, polyvinyl pyrrolidone, or aluminummagnesium silicate. In the usual way, the composition may containadditives other than the inert diluent, such as a lubricant, adisintegrating agent, a stabilizing agent, or a solubilizing orsolubilization assisting agent. If necessary, tablets or pills may becoated with a sugar coating or a film of a gastric or enteric substance.

[0161] The liquid composition for oral administration may include, forexample, emulsions, solutions, suspensions, syrups, and elixirs, and maycontain a generally used inert diluent such as purified water or ethylalcohol. The composition may contain additives other than the inertdiluent, such as moistening agents, suspending agents, sweeteners,flavors, or antiseptics.

[0162] The injections for parenteral administration may include asepticaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof the diluent for use in the aqueous solutions and suspensions includedistilled water for injection use and physiological saline. Examples ofthe diluent for use in the non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, plant oil (e.g., olive oil),alcohols (e.g., ethanol), polysorbate 80 and the like. Such acomposition may further contain a moistening agent, an emulsifyingagent, a dispersing agent, a stabilizing agent, a solubilizing orsolubilization assisting agent, an antiseptic or the like. Thesecompositions may be sterilized, for example, by filtration through abacteria retaining filter, blending of a germicide, or irradiation.Alternatively, they may be used by first making them into sterile solidcompositions and dissolving them in sterile water or other sterilesolvents for injection use, prior to their use.

[0163] The dose is optionally decided by taking into consideration thestrength of each active ingredient selected by the aforementionedscreening method, or symptoms, age, sex, or the like of each patient tobe administered.

[0164] For example, in the case of oral administration, the usual dosagefor an adult (60 kg in weight) is about 0.1 to 100 mg, preferably 0.1 to50 mg per day. In the case of parenteral administration, the usualdosage is about 0.01 to 50 mg, preferably 0.01 to 10 mg per day in theform of an injection.

EXAMPLES

[0165] The present invention now will be further illustrated by, but isby no means limited to, the following Examples. The procedures may beperformed in accordance with the known methods (Maniatis, T., et al.,“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY, 1982, or the like), unless otherwise specified.

Example 1 Isolation of ADP Receptor P2T_(AC) Gene HORK3

[0166] In the present example, a full-length cDNA of an ADP receptorP2T_(AC) gene HORK3 (hereinafter briefly referred to as HORK3 gene) wasobtained by the PCR method using a human brain cDNA (manufactured byClontech) as a template in accordance with the following procedure.

[0167] More particularly, an oligonucleotide consisting of thenucleotide sequence of SEQ ID NO: 3 was used as a forward primer, and anoligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 4was used as a reverse primer. At each of the 5′-termini of the forwardand reverse primers, a nucleotide sequence containing the XbaIrecognition sequence is added. A PCR was carried out using Taq DNAPolymerase (Ex Taq DNA polymerase; manufactured by Takara-shuzo) in thepresence of 5% dimethyl sulfoxide (DMSO). In the PCR, a cycle consistingof treatments at 94° C. for 20 seconds, 58° C. for 20 seconds, and 74°C. for 1.5 minutes was repeated 5 times, a cycle consisting oftreatments at 94° C. for 20 seconds, 55° C. for 20 seconds, and 74° C.for 1.5 minutes was repeated 5 times, and a cycle consisting oftreatments at 94° C. for 20 seconds, 50° C. for 20 seconds, and 74° C.for 1.5 minutes was repeated 25 times. As a result, a DNA fragment ofapproximately 1.0 kbp was amplified. The DNA fragment was digested withXbaI, and inserted into the XbaI site of a plasmid pEF-BOS-dhfr(Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990) toobtain a plasmid pEF-BOS-dhfr-HORK3.

[0168] The nucleotide sequence of the HORK3 gene in the plasmidpEF-BOS-dhfr-HORK3 was determined using a DNA sequencer (ABI377 DNASequencer; manufactured by Applied Biosystems) by a dideoxy terminatormethod. The nucleotide sequence of the HORK3 gene was that of SEQ ID NO:1.

[0169] The nucleotide sequence of SEQ ID NO: 1 contains an open readingframe (ORF) consisting of 1029 bases. The amino acid sequence (342 aminoacids) deduced from the ORF was that of SEQ ID NO: 2.

Example 2 Confirmation of Expression Distribution of HORK3 Gene inHemocytes

[0170] Blood was collected from a healthy volunteer by using heparin,and was centrifuged at 400×g for 10 minutes. The upper layer was takenas platelet-rich plasma.

[0171] To the lower layer, 1/3 volume of 6% dextran/physiological salinewas added, and then the whole was allowed to stand at room temperaturefor 1 hour. The supernatant was taken and centrifuged at 150×g for 5minutes, and then the pellet was suspended in HBSS (Hanks' Balanced SoltSolution). The suspension was layered on an equal volume of Ficoll(Ficoll Paque; Pharmacia), and then the whole was centrifuged at 400×gfor 30 minutes. The resulting intermediate layer and pellet were takenas “a mononuclear cell fraction” and polymorphonuclear leukocytes,respectively.

[0172] CD16 microbeads (manufactured by Daiichi Pure Chemicals) wereadded to the polymorphonuclear leukocytes, and then “a neutrophilfraction” was separated from “an eosinophil fraction” by using amagnetic stand.

[0173] EDTA was added to the previously obtained platelet-rich plasma toa final concentration of 2 mmol/L, and then the whole was centrifuged at2500×g for 15 minutes. The resulting pellet was resuspended in 120mmol/L NaCl/2 mmol/L EDTA/30 mmol/L Tris-HCl (pH7.4), and then thesuspension was centrifuged at 2500×g for 15 minutes to obtain the pelletas “a platelet fraction”. The mononuclear cell fraction, neutrophilfraction, and eosinophil fraction were respectively washed withphysiological saline.

[0174] Total RNAs were purified from the mononuclear cell fraction,neutrophil fraction, eosinophil fraction, and platelet fraction by usinga commercially available total RNA purifying reagent (isogen;manufactured by Nippon Gene), respectively. The total RNA (5 μg) derivedfrom each fraction was reacted with DNase (manufactured by Nippon Gene)at 37° C. for 15 minutes. The DNase-treated total RNA was converted tocDNA by a Superscript first-strand system (for RT-PCR; manufactured byGIBCO).

[0175] An analysis of an expression level of HORK3 mRNA in hemocytes wascarried out by using each of the above cDNAs as a template and asequence detector (Prism7700 Sequence Detector; manufactured by AppliedBiosystems). An oligonucleotide consisting of the base sequence of SEQID NO: 5 and an oligonucleotide consisting of the base sequence of SEQID NO: 6 were used as a primer set. An oligonucleotide consisting of thenucleotide sequence of SEQ ID NO: 7, in which the 5′-terminus thereofwas labeled with a fluorescence indicator FAM (6-carboxy-fluorescein)and the 3′-terminus thereof was labeled with a fluorescence indicatorTAMRA (6-carboxy-tetramethyl-rhodamine), was used as a probe whichspecifically recognizes the HORK3 cDNA.

[0176] The PCR was carried out by using an commercially available PCRreagent (TaqMan PCR core reagent; manufactured by Applied Biosystems)and repeating a cycle consisting of treatments at 95° C. for 15 secondsand 60° C. for 60 seconds 50 times in the presence of 5% DMSO. Further,to obtain a standard curve for calculating an expression level of mRNA,a PCR was carried out under the same conditions by using a human genomicDNA as a template and the above primer set and probe [J. Neurosci.Methods. 98, 9-20 (2000)].

[0177] Furthermore, to calculate, as an internal standard, an expressionlevel of human β-actin, a PCR was carried out under the same conditions,by using the above each cDNA or human genomic DNA as a template, anoligonucleotide consisting of the base sequence of SEQ ID NO: 8 and anoligonucleotide consisting of the base sequence of SEQ ID NO: 9 as aprimer set, and an oligonucleotide consisting of the nucleotide sequenceof SEQ ID NO: 10, in which the 5′-terminus thereof was labeled with afluorescence indicator FAM and the 3′-terminus thereof was labeled witha fluorescence indicator TAMRA, as a probe specifically recognizingβ-actin.

[0178] Expression levels of β-actin mRNA in the mononuclear cellfraction, neutrophil fraction, eosinophil fraction, and plateletfraction were 15000 copies, 19000 copies, 25000 copies, and 33000 copiesper 1 ng of total RNA, respectively. In contrast, it was found thatHORK3 mRNA was specifically expressed in platelets, but little expressedin mononuclear cells, neutrophils, and eosinophils.

Example 3 Confirmation of Increase in Intracellular Ca²⁺ Concentrationby 2MeSADP or ADP in C6-15 Cell Co-expressing HORK3 Protein and Gqi

[0179] When most purines such as ADP, 2MeSADP (2-methylthio-ADP), or thelike are added to a cell, the increase in intracellular Ca²⁺ via anendogenous cellular membrane receptor is observed. Therefore, to analyzewhether or not a protein derived from a gene introduced exogenouslyreacts with ADP or 2MeSADP, it is preferable to express the protein byusing a cell which does not react with ADP or 2MeSADP. It is known thatC6-15, a rat glioma cell line, does not react with ADP or the like(Change, K. et al., J. Biol. Chem., 270, 26152-26158, 1995). In thepresent example, the C6-15 cell was used to express the HORK3 protein.

[0180] Further, the plasmid pEF-BOS-dhfr-HORK3 obtained in Example 1 wasused as an expression plasmid to be used for expressing the HORK3protein.

[0181] An expression vector for expressing the chimeric protein of Gqand Gi used in the present example was prepared in accordance with amethod of Conklin, B. R. et al. (Nature, 363, 274-276, 1993) by cloninga gene (hereinafter referred to as Gqi gene), which had been constructedby substituting five amino acids (Glu-Tyr-Asn-Leu-Val; the amino acidsequence of SEQ ID NO: 12) at the C-terminus of Gq with five amino acids(Asp-Cys-Gly-Leu-Phe; the amino acid sequence of SEQ ID NO: 11) at theC-terminus of Gi, into the plasmid pEF-BOS. The constructed plasmid wasnamed plasmid pEF-BOS-Gqi.

[0182] The C6-15 cells were seeded on a 96-well plate (96 wellBlack/clear bottom plate, collagen I coated; manufactured by BECTONDICKINSON) so that the concentration of cells became 2×10⁴ cells/well.After culturing for 48 hours, a gene transfection was carried out byusing a transfection reagent (LipofectAMINE 2000; manufactured by GIBCOBRL) and the combination of the plasmid pEF-BOS-dhfr-HORK3 (50 ng/well)and the plasmid pEF-BOS-Gqi (50 ng/well). As a control, a genetransfection was carried out by using the combination of the plasmidpEF-BOS-dhfr (i.e., an empty vector without the HORK3 gene) and theplasmid pEF-BOS-Gqi, instead of the combination of the plasmidpEF-BOS-dhfr-HORK3 and the plasmid pEF-BOS-Gqi.

[0183] After 24 hours from the gene transfection procedure, the mediumwas discarded, and then HBBS (Hanks' Balanced Solt Solution; 100μL/well) containing 4 μmol/L Fluo-3, AM (manufactured by MolecularProbe), 0.004% pluronic acid (trademark=Pluronic F127, manufactured byMolecular Probe), 1% fetal bovine serum (FBS), 20 mmol/L HEPES, and 2.5mmol/L probenecid was added. After incubating at 37° C. for 1 hour,cells were washed with HBBS (manufactured by GIBCO) containing 20 mmol/LHEPES and 2.5 mmol/L probenecid 4 times, and then HBBS (100 μL/well)containing 20 mmol/L HEPES and 2.5 mmol/L probenecid was added.

[0184] A time course of the change in the intracellular Ca²⁺concentration was measured by using FLIPR (manufactured by MolecularDevice). More particularly, after 10 seconds from the beginning ofmeasurement, 2MeSADP or ADP was added to wells to a final concentrationof 3×10⁻⁵ mol/L to 1×10⁻¹² mol/L. After the addition, each fluorescenceintensity was measured at an interval of 1 second for 50 seconds, andthen at an interval of 6 seconds for 4 minutes.

[0185] The increase of the intracellular Ca²⁺ concentration dependentupon the concentration of 2MeSADP or ADP was observed in the cells towhich the combination of the plasmid pEF-BOS-dhfr-HORK3 and the plasmidpEF-BOS-Gqi had been transfected. In contrast, the change of theintracellular Ca²⁺ concentration by 2MeSADP or ADP was not observed inthe cells to which the combination of the plasmid pEF-BOS-dhfr (emptyvector) and the plasmid pEF-BOS-Gqi had been transfected.

[0186] The change of the intracellular Ca²⁺ concentration by 2MeSADP orADP in the cells to which the plasmid pEF-BOS-dhfr-HORK3 and the plasmidpEF-BOS-Gqi had been transfected was measured, each peak value invarious concentrations of 2MeSADP or ADP was plotted, and then thedependency upon the concentration was analyzed by using a Logisticregression method. As a result, it was found that EC₅₀ of 2MeSADP was5.4 nmol/L, and that EC₅₀ of ADP was 220 nmol/L. As described above, itwas confirmed that the dose-dependent change of the intracellular Ca²⁺concentration was induced by reacting with 2MeSADP or ADP in thetransformant for Ca²⁺-type detection co-expressing the polypeptide for adetecting tool and Gqi.

[0187] As described above, it was found that the HORK3 protein, one ofthe polypeptides for detection, is a Gi-coupled receptor which reactswith ADP. Further, as apparent from the results in the present example,it has become feasible to screen an agonist or antagonist by measuringthe change of the intracellular Ca²⁺ concentration in the C6-15 cellco-expressing the HORK3 protein (one of the polypeptides for a detectingtool) and Gqi.

Example 4 Confirmation of Inhibition of cAMP Production by 2MeSADP orADP in CHO Cell Expressing HORK3 Protein

[0188] It was found from Example 3 that the ADP receptor was aGi-coupled receptor, and thus it is expected that the ADP receptor hasan activity of suppressing the adenylate cyclase activity. Therefore,when the cAMP-type screening method of the present invention is carriedout, it is preferable to select a cell line not having an activity ofsuppressing the adenylate cyclase activity by ADP or 2MeSADP, as a hostcell to be used for expressing the ADP receptor protein. It was foundthat the CHO cell was the most preferable by searching for a cell inwhich an amount of cAMP produced by a forskolin stimulus was notdecreased by ADP or 2MeSADP in accordance with the following procedure,and thus the CHO cell was used as the cell to be used for expressing theADP receptor protein. In this connection, a dihydrofolate reductase(DHFR; an essential enzyme for de novo synthesis of nucleic acids)defective cell line [CHO-dhfr(−) line] was used in the present example.The plasmid pEF-BOS-dhfr-HORK3 was used as an expression plasmid to beused for expressing the HORK3 protein.

[0189] The CHO-dhfr(−) line was seeded on each 10 cm-petri dish (1×10⁶cells/dish) by using an αMEM (with nucleic acids) medium. Afterculturing for 1 day, a gene transfection was carried out by using atransfection reagent (LipofectAMINE 2000; manufactured by GIBCO BRL) andthe plasmid pEF-BOS-dhfr-HORK3 (8 μg). As a control, a gene transfectionwas carried out by using the plasmid pEF-BOS-dhfr (i.e., an empty vectorwithout the HORK3 gene), instead of the plasmid pEF-BOS-dhfr-HORK3.

[0190] After 24 hours from the gene transfection procedure, thetransfected cells were taken and suspended in an αMEM (without nucleicacids) medium containing 100 nmol/L methotrexate (a competitiveinhibitor of DHFR; manufactured by Wako Pure Chemical Industries). Eachsuspension was gradually diluted and reseeded on each 10 cm-petri dish.Colonies which appeared after 2 weeks were individually taken, and usedas a CHO cell expressing the HORK3 protein or a control CHO celltransfected with the empty vector in the following experiment.

[0191] The CHO cells expressing the HORK3 protein or the CHO cellstransfected with the empty vector were seeded on a 24-well plate (1×10⁵cells/well). After culturing for 1 day, cells were treated with an αMEM(without nucleic acids) medium containing 1 mmol/L3-isobutyl-1-methylxanthine (manufactured by Sigma) and 0.1% BSA for 10minutes, and then the combination of 3 μmol/L forskolin (manufactured byWako Pure Chemical Industries) and 2MeSADP (a finalconcentration=1×10⁻¹² to 1×10⁻⁷ mol/L), or the combination of 3 μmol/Lforskolin and ADP (a final concentration=1×10⁻¹⁰ to 1×10⁻⁵ mol/L) wasadded dropwise. After incubating at 37° C. for 30 minutes, the culturesupernatant was discarded, and then cells were dissolved in a cell lysisreagent contained in a cAMP-EIA system (BIOTRAK; manufactured byAmersham).

[0192] The amount of cAMP produced in each cell under each condition wasmeasured by using the cAMP-EIA system in accordance with a protocolattached thereto. When the amount of cAMP produced by stimulating with 3μmol/L forskolin alone was regarded as 100%, a concentration-dependentcurve for the amount of cAMP in the presence of 2MeSADP or ADP wasdrawn. From the concentration-dependent curve, the responsiveness of2MeSADP and ADP against the HORK3 protein were EC₅₀=0.07 nmol/L andEC₅₀=35 nmol/L, respectively.

[0193] In contrast, no changes in the amount of cAMP produced by theforskolin stimulus were observed in the CHO cell transfected with theempty vector, even if 2MeSADP or ADP was added.

Example 5 Effects of Inhibitors and Confirmation of Inhibition of cAMPProduction by 2MeSADP or ADP in C6-15 Cell Expressing HORK3 Protein

[0194] It was found that the C6-15 cell was also the most preferable asa cell in which the amount of cAMP produced by the forskolin stimuluswas not decreased by ADP or 2MeSADP, and thus the C6-15 cell was alsoused as the cell to be used for expressing the ADP receptor protein.

[0195] The plasmid pEF-BOS-dhfr-HORK3 was used as an expression vectorto be used for expressing the HORK3 protein.

[0196] The C6-15 cells were seeded on each 10 cm-petri dish (1×10⁶cells/dish) by using a DMEM medium. After culturing for 1 day, an genetransfection was carried out by using a transfection reagent(LipofectAMINE 2000; manufactured by GIBCO BRL), and the plasmidspEF-BOS-dhfr-HORK3 (8 μg) and pEF-BOS-neo (Nucleic Acid Res., 18, 5322,1990; 0.8 μg).

[0197] After 24 hours from the gene transfection procedure, thetransfected cells were taken and suspended in a DMEM medium containing0.6 mg/mL G418 (manufactured by GIBCO BRL). Each suspension wasgradually diluted and reseeded on each 10 cm-petri dish. Colonies whichappeared after 2 weeks were individually taken, and used as a C6-15 cellexpressing the HORK3 protein in the following experiment.

[0198] The C6-15 cells expressing the HORK3 protein were seeded on a96-well plate (1×10⁴ cells/well). After culturing for 1 day, cells weretreated with a DMEM medium containing 1 mmol/L3-isobutyl-1-methylxanthine (manufactured by Sigma) and 0.1% BSA for 10minutes, and then the combination of 1 μmol/L forskolin (manufactured byWako Pure Chemical Industries) and 2MeSADP (a finalconcentration=1×10⁻¹² to 1×10⁻⁷ mol/L), or the combination of 1 μmol/Lforskolin and ADP (a final concentration=1×10⁻¹⁰ to 1×10⁻⁵ mol/L) wasadded dropwise. After incubating at 37° C. for 30 minutes, the culturesupernatant was discarded, and then cells were dissolved in PBScontaining 0.2% Triton X-100.

[0199] The amount of cAMP produced in each cell under each condition wasmeasured by using a cAMP HTRF kit (manufactured by CIS biointernational) in accordance with a protocol attached thereto. When theamount of cAMP produced by stimulating with 1 μmol/L forskolin alone wasregarded as 100%, a concentration-dependent curve for the amount of cAMPin the presence of 2MeSADP or ADP was drawn. From theconcentration-dependent curve, the responsiveness of 2MeSADP and ADPagainst the HORK3 protein were EC₅₀=0.08 nmol/L and EC₅₀=42 nmol/L,respectively. The C6-15 cell expressing the HORK3 protein exhibitedalmost the same property as that of the CHO cell expressing the HORK3protein described in Example 4.

[0200] Next, effects of compounds, which are known to suppress theplatelet aggregation, on the suppression of the amount of cAMP producedby the forskolin stimulus in the C6-15 cell expressing the HORK3 proteinby 2MeSADP were examined. As the compounds suppressing the plateletaggregation, 2MeSAMP (2-methylthio-adenosine monophosphate) (Thromb.Haemost., 81, 111-117, 1999) or AR-C69931MX(N6-[2-methylthioethyl]-2-[3,3,3-trifluoropropylthio]-5′-ade nylic acid,monoanhydride with dichloromethylenebiphosphonic acid) (J. Med. Chem.,42, 213-220, 1999) was used. In the above measurement system, cells weretreated with a solution [prepared by dissolving 2MeSAMP (10⁻⁷ to 10⁻⁴mol/L) or AR-C69931MX (10⁻¹² to 10⁻⁶ mol/L) in DMEM medium containing 1mmol/L 3-isobutyl-1-methylxanthine (manufactured by Sigma) and 0.1% BSA]for 10 minutes, and then the combination of 1 μmol/L forskolin(manufactured by Wako Pure Chemical Industries) and 10 nmol/L 2MeSADPwas added dropwise. After incubating at 37° C. for 30 minutes, theculture supernatant was discarded, and then cells were dissolved in PBScontaining 0.2% Triton X-100. The amount of cAMP produced was measuredby using the cAMP HTRF kit in accordance with a protocol attachedthereto. When the amount of cAMP produced by stimulating with 1 μmol/Lforskolin alone was regarded as 0%, and that by stimulating with thecombination of 1 μmol/L forskolin and 10 nmol/L 2MeSADP was regarded as100%, a concentration-dependent curve of each inhibitor was drawn. Fromthe concentration-dependent curve, inhibitory effects of 2MeSAMP andAR-C69931MX on the suppression by 2MeSADP in the amount of cAMP producedby the forskolin stimulus were IC₅₀=5 μmol/L and IC₅₀=2.4 nmol/L,respectively.

Example 6 Binding Assay of 2MeSADP to C6-15 Cell Expressing HORK3Protein

[0201] The C6-15 cells expressing the HORK3 protein prepared in Example5 were taken and washed, and then suspended in 20 mmol/L Tris-HCl(pH7.4) containing 5 mmol/L EDTA and a protease inhibitor cocktail set(Complete™; manufactured by Boeringer Mannheim). The whole washomogenized by using a POLYTRON, and ultracentrifuged. The pellet wassuspended in 50 mmol/L Tris-HCl (pH7.4) containing 1 mmol/L EDTA, 100mmol/L NaCl, 0.1% BSA, and Complete™ to obtain a membrane fraction.

[0202] After [³H]-2MeSADP (manufactured by Moravek Biochemical) wasadded to 20 μg of the membrane fraction as a final concentration of 0.25to 50 nmol/L, and was incubated in 250 μL of 50 mmol/L Tris-HCl (pH7.4)containing 1 mmol/L EDTA, 100 mmol/L NaCl, 0.1% BSA, and Complete™ atroom temperature for 1 hour, the whole was collected on a glass filterby using a cell harvester. A microscintillator was added to the glassfilter, and then a total amount of binding to the membrane fraction wasmeasured by using a liquid scintillation counter. Further, an amount ofnon-specific binding to the membrane fraction was measured by adding2MeSADP (a final concentration=50 μmol/L) in the above assay. As aresult, it was found that [³H]-2MeSADP was specifically bound to themembrane fraction of the C6-15 cell expressing the HORK3 protein. As aresult of the Scatchard analysis for the binding, the dissociationconstant of the [³H]-2MeSADP binding to the membrane fraction of theC6-15 cell expressing the HORK3 protein was Kd=0.27 nmol/L, and themaximum binding was Bmax=3.7 pmol/mg. In contrast, such a specificbinding was not observed in the membrane fraction of the host cell C6-15not expressing the HORK3 protein.

[0203] Next, an effect of 2MeSAMP or AR-C69931MX was examined by usingthe membrane fraction of the C6-15 cell expressing the HORK3 proteinprepared in Example 6, and by using an activity of inhibiting the[³H]-2MeSADP binding as an indicator. More particularly, after 2MeSAMP(10⁻⁷ to 10⁻⁴ mol/L) or AR-C69931MX (10⁻¹² to 10⁻⁶ mol/L) and 1 nmol/L[³H]-2MeSADP were added to 20 μg of the membrane fraction from the C6-15cell expressing the HORK3 protein, and were incubated at roomtemperature for 2 hours, the whole was collected on a glass filter byusing a cell harvester. A microscintillator was added to the glassfilter, and then radioactivity was measured by using a liquidscintillation counter. Further, radioactivity was measured by not addingthe compound and by adding 2MeSADP (a final concentration=10 μmol/L) inthe above assay, as a total amount of binding and an amount ofnon-specific binding, respectively. From the concentration-dependentcurve of each compound, the inhibitory effects of 2MeSAMP andAR-C69931MX on the binding of 2MeSADP to the HORK3 protein were IC₅₀=4.9μmol/L and IC₅₀=9.8 nmol/L, respectively.

[0204] In addition to the results in Example 3, the amount of cAMPproduced by the forskolin stimulus in the CHO and C6-15 cells expressingthe HORK3 protein was suppressed by 2MeSADP or ADP in Examples 4 and 5,respectively. These results made it clearer that the ADP receptor wascoupled to Gi. Further, from the results in Examples 4 and 5, it hasbecome feasible to screen an agonist or antagonist by measuring thechange of the intracellular cAMP concentration in the CHO or C6-15 cellexpressing the HORK3 protein (one of the polypeptides for a screeningtool). Furthermore, it is possible to screening a ligand by measuringthe inhibition of 2MeSADP binding to the membrane fraction of the C6-15cell expressing the HORK3 protein, in accordance with the methoddescribed in Example 6.

[0205] As described above, the HORK3 protein is a Gi-coupled receptorexpressed in platelets, from the results in Examples 2 to 6, and thus itis considered that the HORK3 protein is an entity of the platelet ADPreceptor P2T_(AC) in which the existence thereof has been suggested.

Example 7 Confirmation of Activity of Inhibiting Human PlateletAggregation

[0206] Blood was collected from a healthy person (adult, male) by using1/10 volume of sodium citrate, and then platelet-rich plasma(PRP) wasprepared in accordance with a method of De Marco et al. (J. Clin.Invest., 77, 1272-1277, 1986). The PRP was used after preparing it to3×10⁸ cells/mL by using an automatic blood cell counter (MEK6258; NihonKohden Corporation). As an inducer of aggregation, ADP which is aproduct manufactured by MC Medical Corporation was used. Further,2MeSAMP or AR-C69931MX was used, and physiological saline was used as asolvent to be used for dissolving the compounds. In this connection, itwas confirmed in Example 5 that 2MeSAMP or AR-C69931MX exhibited anactivity as a HORK3 protein antagonist.

[0207] The platelet aggregation was measured by using an aggregometer(MCM Hema Tracer 212; MC Medical Corpration). More particularly, PRP (80μL) and a sample (2MeSAMP or AR-C69931MX) or the solvent (10 μL) wereincubated at 37° C. for 1 minute, and then 10 μL of ADP (20 to 200μmol/L) was added. Changes of a transmitted light were recorded for 10minutes, and then an aggregation inhibition (%) was calculated from themaximum aggregation rate.

[0208] The results when 2MeSAMP and AR-C69931MX were used are shown inFIGS. 1 and 2, respectively. The data for 2MeSAMP (FIG. 1) arerepresented by the “average” of the experiment results obtained by usingtwo volunteers. The data for AR-C69931MX (FIG. 2) are represented by the“average+standard error” of the experiment results obtained by usingthree volunteers. Both agents inhibited the ADP-induced plateletaggregation concentration-dependently. The inhibitory strength wasdependent on the concentration of ADP as an inducer.

[0209] It is apparent from the present example that an HORK3 proteinantagonist exhibits an antiplatelet activity.

INDUSTRIAL APPLICABILITY

[0210] A substance inhibiting the platelet ADP receptor P2T_(AC)activity exhibits an antiplatelet activity, and makes it possible totreat a thrombotic disorders.

[0211] Therefore, according to the screening tool of the presentinvention for an antiplatelet agent, it is possible to screen andevaluate a platelet ADP receptor P2T_(AC) antagonist which is useful asan antiplatelet agent.

[0212] It is possible to select a platelet ADP receptor P2T_(AC)antagonist and to screen a substance which is useful as an antiplateletagent, by using the detecting method of the present method for anantagonist or agonist, or by using the combination of the liganddetecting method of the present invention and the detecting method ofthe present method for an antagonist or agonist.

[0213] Further, it is possible to manufacture a pharmaceuticalcomposition for antiplatelet by preparing a medicament by using thesubstance selected by the above screening method as an activeingredient, and carriers, fillers, and/or other additives.

[0214] Furthermore, it is possible to use the detecting method of thepresent invention for a ligand, antagonist, or agonist not only forscreening the substance useful as an antiplatelet agent, but also as aquality control test of a pharmaceutical composition for antiplatelet.

[0215] It is possible to manufacture a pharmaceutical composition forantiplatelet, by detecting whether or not a test compound is a plateletADP receptor P2T_(AC) ligand, antagonist, or agonist using the detectingmethod of the present invention for a ligand, antagonist, or agonist,and then by preparing a medicament using the antagonist or ligand.

Free Text in Sequence Listing

[0216] Features of “Artificial Sequence” are described in the numericidentifier <223> in the Sequence Listing. More particularly, eacholigonucleotide consisting of the base sequence of SEQ ID NO: 3 or 4 isan artificially synthesized primer sequence.

[0217] As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1 12 1 1029 DNA Homo sapiens CDS (1)..(1029) 1 atg caa gcc gtc gac aacctc acc tct gcg cct ggg aac acc agt ctg 48 Met Gln Ala Val Asp Asn LeuThr Ser Ala Pro Gly Asn Thr Ser Leu 1 5 10 15 tgc acc aga gac tac aaaatc acc cag gtc ctc ttc cca ctg ctc tac 96 Cys Thr Arg Asp Tyr Lys IleThr Gln Val Leu Phe Pro Leu Leu Tyr 20 25 30 act gtc ctg ttt ttt gtt ggactt atc aca aat ggc ctg gcg atg agg 144 Thr Val Leu Phe Phe Val Gly LeuIle Thr Asn Gly Leu Ala Met Arg 35 40 45 att ttc ttt caa atc cgg agt aaatca aac ttt att att ttt ctt aag 192 Ile Phe Phe Gln Ile Arg Ser Lys SerAsn Phe Ile Ile Phe Leu Lys 50 55 60 aac aca gtc att tct gat ctt ctc atgatt ctg act ttt cca ttc aaa 240 Asn Thr Val Ile Ser Asp Leu Leu Met IleLeu Thr Phe Pro Phe Lys 65 70 75 80 att ctt agt gat gcc aaa ctg gga acagga cca ctg aga act ttt gtg 288 Ile Leu Ser Asp Ala Lys Leu Gly Thr GlyPro Leu Arg Thr Phe Val 85 90 95 tgt caa gtt acc tcc gtc ata ttt tat ttcaca atg tat atc agt att 336 Cys Gln Val Thr Ser Val Ile Phe Tyr Phe ThrMet Tyr Ile Ser Ile 100 105 110 tca ttc ctg gga ctg ata act atc gat cgctac cag aag acc acc agg 384 Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg TyrGln Lys Thr Thr Arg 115 120 125 cca ttt aaa aca tcc aac ccc aaa aat ctcttg ggg gct aag att ctc 432 Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu LeuGly Ala Lys Ile Leu 130 135 140 tct gtt gtc atc tgg gca ttc atg ttc ttactc tct ttg cct aac atg 480 Ser Val Val Ile Trp Ala Phe Met Phe Leu LeuSer Leu Pro Asn Met 145 150 155 160 att ctg acc aac agg cag ccg aga gacaag aat gtg aag aaa tgc tct 528 Ile Leu Thr Asn Arg Gln Pro Arg Asp LysAsn Val Lys Lys Cys Ser 165 170 175 ttc ctt aaa tca gag ttc ggt cta gtctgg cat gaa ata gta aat tac 576 Phe Leu Lys Ser Glu Phe Gly Leu Val TrpHis Glu Ile Val Asn Tyr 180 185 190 atc tgt caa gtc att ttc tgg att aatttc tta att gtt att gta tgt 624 Ile Cys Gln Val Ile Phe Trp Ile Asn PheLeu Ile Val Ile Val Cys 195 200 205 tat aca ctc att aca aaa gaa ctg taccgg tca tac gta aga acg agg 672 Tyr Thr Leu Ile Thr Lys Glu Leu Tyr ArgSer Tyr Val Arg Thr Arg 210 215 220 ggt gta ggt aaa gtc ccc agg aaa aaggtg aac gtc aaa gtt ttc att 720 Gly Val Gly Lys Val Pro Arg Lys Lys ValAsn Val Lys Val Phe Ile 225 230 235 240 atc att gct gta ttc ttt att tgtttt gtt cct ttc cat ttt gcc cga 768 Ile Ile Ala Val Phe Phe Ile Cys PheVal Pro Phe His Phe Ala Arg 245 250 255 att cct tac acc ctg agc caa acccgg gat gtc ttt gac tgc act gct 816 Ile Pro Tyr Thr Leu Ser Gln Thr ArgAsp Val Phe Asp Cys Thr Ala 260 265 270 gaa aat act ctg ttc tat gtg aaagag agc act ctg tgg tta act tcc 864 Glu Asn Thr Leu Phe Tyr Val Lys GluSer Thr Leu Trp Leu Thr Ser 275 280 285 tta aat gca tgc ctg gat ccg ttcatc tat ttt ttc ctt tgc aag tcc 912 Leu Asn Ala Cys Leu Asp Pro Phe IleTyr Phe Phe Leu Cys Lys Ser 290 295 300 ttc aga aat tcc ttg ata agt atgctg aag tgc ccc aat tct gca aca 960 Phe Arg Asn Ser Leu Ile Ser Met LeuLys Cys Pro Asn Ser Ala Thr 305 310 315 320 tct ctg tcc cag gac aat aggaaa aaa gaa cag gat ggt ggt gac cca 1008 Ser Leu Ser Gln Asp Asn Arg LysLys Glu Gln Asp Gly Gly Asp Pro 325 330 335 aat gaa gag act cca atg taa1029 Asn Glu Glu Thr Pro Met 340 2 342 PRT Homo sapiens 2 Met Gln AlaVal Asp Asn Leu Thr Ser Ala Pro Gly Asn Thr Ser Leu 1 5 10 15 Cys ThrArg Asp Tyr Lys Ile Thr Gln Val Leu Phe Pro Leu Leu Tyr 20 25 30 Thr ValLeu Phe Phe Val Gly Leu Ile Thr Asn Gly Leu Ala Met Arg 35 40 45 Ile PhePhe Gln Ile Arg Ser Lys Ser Asn Phe Ile Ile Phe Leu Lys 50 55 60 Asn ThrVal Ile Ser Asp Leu Leu Met Ile Leu Thr Phe Pro Phe Lys 65 70 75 80 IleLeu Ser Asp Ala Lys Leu Gly Thr Gly Pro Leu Arg Thr Phe Val 85 90 95 CysGln Val Thr Ser Val Ile Phe Tyr Phe Thr Met Tyr Ile Ser Ile 100 105 110Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg Tyr Gln Lys Thr Thr Arg 115 120125 Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu Leu Gly Ala Lys Ile Leu 130135 140 Ser Val Val Ile Trp Ala Phe Met Phe Leu Leu Ser Leu Pro Asn Met145 150 155 160 Ile Leu Thr Asn Arg Gln Pro Arg Asp Lys Asn Val Lys LysCys Ser 165 170 175 Phe Leu Lys Ser Glu Phe Gly Leu Val Trp His Glu IleVal Asn Tyr 180 185 190 Ile Cys Gln Val Ile Phe Trp Ile Asn Phe Leu IleVal Ile Val Cys 195 200 205 Tyr Thr Leu Ile Thr Lys Glu Leu Tyr Arg SerTyr Val Arg Thr Arg 210 215 220 Gly Val Gly Lys Val Pro Arg Lys Lys ValAsn Val Lys Val Phe Ile 225 230 235 240 Ile Ile Ala Val Phe Phe Ile CysPhe Val Pro Phe His Phe Ala Arg 245 250 255 Ile Pro Tyr Thr Leu Ser GlnThr Arg Asp Val Phe Asp Cys Thr Ala 260 265 270 Glu Asn Thr Leu Phe TyrVal Lys Glu Ser Thr Leu Trp Leu Thr Ser 275 280 285 Leu Asn Ala Cys LeuAsp Pro Phe Ile Tyr Phe Phe Leu Cys Lys Ser 290 295 300 Phe Arg Asn SerLeu Ile Ser Met Leu Lys Cys Pro Asn Ser Ala Thr 305 310 315 320 Ser LeuSer Gln Asp Asn Arg Lys Lys Glu Gln Asp Gly Gly Asp Pro 325 330 335 AsnGlu Glu Thr Pro Met 340 3 34 DNA Artificial Sequence Description ofArtificial Sequencean artificially synthesized primer sequence 3cctctagaat gcaagccgtc gacaacctca cctc 34 4 34 DNA Artificial SequenceDescription of Artificial Sequencean artificially synthesized primersequence 4 cctctagact attacattgg agtctcttca tttg 34 5 17 DNA Homosapiens 5 gcaagccgtc gacaacc 17 6 24 DNA Homo sapiens 6 tgattttgtagtctctggtg caca 24 7 24 DNA Homo sapiens 7 cacctctgcg cctggtaaca ccag 248 17 DNA Homo sapiens 8 cactgagcgc ggctaca 17 9 22 DNA Homo sapiens 9cttaatgtca cgcacgattt cc 22 10 20 DNA Homo sapiens 10 cttcaccaccacggccgagc 20 11 5 PRT Homo sapiens 11 Asp Cys Gly Leu Phe 1 5 12 5 PRTHomo sapiens 12 Glu Tyr Asn Leu Val 1 5

1. A screening tool for an antiplatelet agent, wherein the tool is (1) apolypeptide having an amino acid sequence of SEQ ID NO: 2, or (2) apolypeptide having an amino acid sequence in which one or plural aminoacids are deleted, substituted, and/or added at one or plural positionsin an amino acid sequence of SEQ ID NO: 2, and exhibiting an activity ofsuppressing an adenylate cyclase activity by binding to ADP and couplingwith Gi.
 2. The screening tool for an antiplatelet agent according toclaim 1, wherein the tool is a polypeptide having an amino acid sequenceof SEQ ID NO:
 2. 3. A screening tool for an antiplatelet agent, whereinthe tool is a polypeptide having an amino acid sequence having a 90% ormore homology with an amino acid sequence of SEQ ID NO: 2, andexhibiting an activity of suppressing an adenylate cyclase activity bybinding to ADP and coupling with Gi.
 4. The screening tool for anantiplatelet agent according to claim 3, wherein the tool is apolypeptide having an amino acid sequence of SEQ ID NO:
 2. 5. Ascreening tool for an antiplatelet agent, wherein the tool is atransformant which is transformed with an expression vector comprising aDNA encoding the polypeptide according to claim 1 and is expressing thepolypeptide.
 6. The screening tool for an antiplatelet agent accordingto claim 5, wherein the tool is a transformant which is transformed withan expression vector comprising a DNA encoding a polypeptide having anamino acid sequence of SEQ ID NO: 2 and is expressing the polypeptide.7. A screening tool for an antiplatelet agent, wherein the tool is atransformant which is transformed with an expression vector comprising aDNA encoding the polypeptide according to claim 3 and is expressing thepolypeptide.
 8. The screening tool for an antiplatelet agent accordingto claim 7, wherein the tool is a transformant which is transformed withan expression vector comprising a DNA encoding a polypeptide having anamino acid sequence of SEQ ID NO: 2 and is expressing the polypeptide.9. A method for detecting whether or not a compound to be tested is anADP receptor P2T_(AC) ligand, comprising the steps of: bringing apolypeptide according to claim 1 or 3, a cell membrane fractioncomprising the polypeptide, or a transformant according to claim 5 or 7into contact with the compound to be tested, in the presence of alabeled ligand of an ADP receptor P2T_(AC); and analyzing a change of anamount of the labeled ligand which binds to the polypeptide, the cellmembrane fraction, or the transformant.
 10. A method for detectingwhether or not a compound to be tested is an ADP receptor P2T_(AC)antagonist or agonist, comprising the steps of: bringing the compound tobe tested into contact with a transformant according to claim 5 or 7which is co-expressing a G protein chimera comprising a polypeptidefragment having an activity promoting a phospholipase C activity of a Gprotein promoting the phospholipase C activity and a polypeptidefragment having a receptor-coupled activity of Gi, said G proteinchimera having an amino acid sequence of SEQ ID NO: 11 at theC-terminus; and analyzing a change of an intracellular Ca²⁺concentration in the transformant.
 11. A method for detecting whether ornot a compound to be tested is an ADP receptor P2T_(AC) antagonist oragonist, comprising the steps of: bringing a transformant according toclaim 5 or 7 into contact with the compound to be tested, in thepresence of a platelet ADP receptor P2T_(AC) agonist; and analyzing achange of an intracellular cAMP concentration in the transformant.
 12. Amethod for screening an antiplatelet agent, comprising the steps of:detecting whether or not a compound to be tested is an ADP receptorP2T_(AC) ligand, antagonist, or agonist by the method according to anyone of claims 9 to 11 or a combination thereof; and selecting the ADPreceptor P2T_(AC) antagonist.
 13. A process for manufacturing apharmaceutical composition for antiplatelet, comprising the steps of:detecting whether or not a compound to be tested is an ADP receptorP2T_(AC) ligand, antagonist, or agonist by the method according to anyone of claims 9 to 11 or a combination thereof; and preparing amedicament.